This project is studying assembly and degradation of myofibrillar proteins in muscle with emphasis on the role of Z-disk and M-line proteins in these processes. Specific goals of this project are: 1) to determine the molecular architecture of the Z-disk and the proteins constituting the Z-disk; 2) to ascertain the properties and physiological role of alpha-actinin, a Z-disk protein; 3) to purify and characterize the proteins constituting the M-line; and 4) to determine when in muscle cell differentiation Z-disk and M-line proteins are first synthesized and assembled, the role of Z-disk ad M-line proteins in assembly of myofibrillar proteins, and the mechanism of degradation of intact myofibrils. The molecular architecture and protein constitution of the Z-disk is being studied by using antibodies against purified alpha-actinin, tropomyosin, and actin in conjunction with selective extraction by low ionic strength solvents and proteolytic digestion by a highly specific protease isolated from skeletal muscle. The properties and physiological role of alpha-actinin is being studied by using proteolytic enzymes as structural probes, by localization of alpha- actinin with alpha-actinin antibodies, and by examining the effect of alpha-actinin on conformation of actin. M-line proteins are being studied by using selective extraction and by antibody localization techniques. The stage in cellular differentiation that M-line and Z- disk proteins are first synthesized and assembled are studied by using cell cultures, rate of incorporation of radioactive amino acids into purified myofibrillar proteins, and by measuring the binding of antibodies against purified Z-disk and M-line proteins to myogenic cells in various stages of differentiation.